The composition of a plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various .beta.-xylan chains) and pectin. The occurrence, distribution and structural features of plant cell wall polysaccharides are determined by (1) plant species; (2) variety; (3) tissue type, (4) growth conditions; (5) aging and (6) processing of plant material prior to feeding.
Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant (Chesson, 1987; Carre and Brillouet, 1986). Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone. The main structure of hemicellulose in dicotyledons is a xyloglucan complex. Moreover, higher pectin concentrations are found in dicotyledons than in monocotyledons. Seeds are generally very high in pectic substances but relatively low in cellulosic material. Three more or less interacting polysaccharide structures can be distinguished in the cell wall:
(1) The middle lamella forms the exterior cell wall. It also serves as the point of attachment for the individual cells to one another within the plant tissue matrix. The middle lamella consists primarily of calcium salts of highly esterified pectins; PA1 (2) The primary wall is situated just inside the middle lamella. It is a well-organized structure of cellulose microfibrils embedded in an amorphous matrix of pectin, hemicellulose, phenolic esters and proteins; PA1 (3) The secondary wall is formed as the plant matures. During the plant's growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited.
The primary cell wall of mature, metabolically active plant cells (e.g. mesophyll and epidermis) is more susceptible to enzymatic hydrolysis than the secondary cell wall, which by this stage, has become highly lignified.
There is a high degree of interaction between cellulose, hemicellulose and pectin in the cell wall. The enzymatic degradation of these rather intensively cross-linked polysaccharide structures is not a simple process. At least five different enzymes are needed to completely break down an arabinoxylan, for example. The endo-cleavage is effected by the use of an endo-.crclbar.(1.fwdarw.4)-D-xylanase. Exo-(1.fwdarw.4)-D-xylanase liberates xylose units at the non-reducing end of the polysaccharide. Three other enzymes (.alpha.-glucuronidase, .alpha.-L-arabinofuranosidase and acetyl esterase) are used to attack substituents on the xylan backbone. The choice of the specific enzymes is of course dependent on the specific hemicellulose to be degraded (McCleary and Matheson, 1986).
For certain applications, however, complete degradation of the entire hemicellulose into monomers is not necessary or is not desirable. In the liquefaction of arabinoxylan, for example, one needs simply to cleave the main xylan backbone into shorter units. This may be achieved by the action of an endo-xylanase, which ultimately results in a mixture of xylose monomer units and oligomers such as xylobiose and xylotriose. These shorter subunits are then sufficiently soluble for the desired use. Furthermore, it has been demonstrated that the actions of specific xylanase enzymes differ from one another, as seen by the varying patterns of xylose monomer and oligomer units resulting from the action of these specific enzymes on an arabinoxylan substrate (Kormelink, F., 1992).
Filamentous fungi are widely known for their capacity to secrete large amounts of a variety of hydrolytic enzymes such as .alpha.-amylases, proteases and amyloglucosidases and various plant cell wall degrading enzymes such as cellulases, hemicellulases, and pectinases. Among these, multiple xylan-degrading enzymes have been recognized, which have been shown to possess a variety of biochemical and physical properties. This heterogeneity in xylanase function allows for the selection of a xylanase of interest which is best suited for a desired application (see Wong et al. (1988), Woodward (1984) and Dekker and Richards (1977)).
Multiple xylanases of various molecular weights are known to be produced by micro-organisms such as Aspergillus niger, Aspergillus tubigensis, Clostridium thermocellum, Trichoderma reesei, Penicillium janthinellum, as well as species of Bacillus and Streptomyces. In Aspergillus tubigensis, for example, three distinct xylanases (XYL A, B and C) have been identified.
In nature, microbial xylanases are always produced together with other enzymes having polysaccharide-degrading activities, such as exo-arabinanase, acetyl esterase and cellulases. For some applications, such as the bleaching of lignocellulosic pulp, some of these enzyme activities are not needed or are unwanted.
It is known that fermentation conditions may be varied to favor the production of an enzyme of interest. It is also known that the cloning of the gene encoding the desired enzyme and overexpressing it in its natural host, or other compatible expression host will specifically enhance the production of the enzyme of interest. This latter method is particularly useful if the enzyme of interest is to be obtained in a form which is free of undesired enzyme activity.
The cloning of the A. tubigensis gene encoding the xylanase A (XYL A) enzyme has been described by Van den Broeck et al. (1992). The description is hereby incorporated by reference.
However it has been found that the A. tubigensis XYL B enzyme has slightly higher pH and temperature optima than does the XYL A enzyme. For certain applications such as the bleaching of lignocellulosic pulp, xylanases having higher pH and temperature optima are preferred, particularly those xylanase enzyme preparations having little or no cellulase activity.
Unfortunately, A. tubigensis produces the XYL B enzyme in lower quantities than either the XYL A or XYL C proteins. This coupled with the fact that the XYL B enzyme is difficult to purify from the other enzymes in the culture broth from the fermentation of A. tubigensis make the production of XYL B via classical fermentation and purification techniques economically unfeasible.
Efforts to clone the xlnB gene (which encodes the XYL B enzyme) from A. tubigensis using the protocol for hybridization to the xylA gene as described by van den Broeck et al. (1992) failed to yield a positive signal. This implies that it is not possible to isolate the xlnB gene by heterologous hybridization to the xlnA gene.
Nonetheless, it would be of great importance to obtain genes encoding an enzyme having the activity of the A. tubigensis XYL B enzyme which may be brought to expression in other, high-producing microbial expression hosts. In this manner, the production and use of an enzyme having the activity of the A. tubigensis XYL B enzyme (optionally lacking the presence of undesirable side activities may be made economically feasible for industrial applications.